US6656922B2 - Oral delivery of macromolecules - Google Patents

Oral delivery of macromolecules Download PDF

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US6656922B2
US6656922B2 US09/845,827 US84582701A US6656922B2 US 6656922 B2 US6656922 B2 US 6656922B2 US 84582701 A US84582701 A US 84582701A US 6656922 B2 US6656922 B2 US 6656922B2
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acid
heparin
doca
mixtures
agent
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US20020010153A1 (en
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Youngro Byun
Yong-kyu Lee
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Mediplex Corp Korea
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Mediplex Corp Korea
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Priority claimed from US09/300,173 external-priority patent/US6245753B1/en
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Assigned to MEDIPLEX CORPORATION reassignment MEDIPLEX CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BYUN, YOUNGRO, LEE, YONG-KYU
Priority to JP2002584942A priority patent/JP2004532851A/ja
Priority to EP01976911A priority patent/EP1383518A4/en
Priority to CNA018231993A priority patent/CN1518452A/zh
Priority to PCT/KR2001/001723 priority patent/WO2002087597A1/en
Priority to KR1020020000622A priority patent/KR20020083905A/ko
Publication of US20020010153A1 publication Critical patent/US20020010153A1/en
Priority to US10/727,078 priority patent/US20040220143A1/en
Publication of US6656922B2 publication Critical patent/US6656922B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof

Definitions

  • This invention relates to derivatives of macromolecules, including polysaccharide derivatives, having increased hydrophobicity as compared to the unmodified macromolecules or polysaccharides. More particularly, the invention relates to oral delivery and absorption of hydrophobized macromolecules and amphiphilic polysaccharide derivatives, such as amphiphilic heparin derivatives, wherein the bioactivity of the macromolecule or polysaccharide is preserved.
  • the hydrophobized macromolecules and amphiphilic polysaccharide derivatives have a molecular weight of greater than 1000, yet are absorbed after oral administration.
  • Heparin is a polysaccharide composed of sulfated D-glucosamine and D-glucuronic acid residues. Due to its numerous ionizable sulfate groups, heparin possesses a strong electronegative charge. It is also a relatively strong acid that readily forms water-soluble salts, e.g. heparin sodium. It is found in mast cells and can be extracted from many body organs, particularly those with abundant mast cells. The liver and lungs are especially rich in heparin. The circulating blood contains no heparin except after profound disruption of mast cells. Heparin has many physiological roles, such as blood anticoagulation, inhibition of smooth muscle cell proliferation, and others.
  • heparin is a potent anticoagulant agent that interacts strongly with antithrombin III (ATIII) to prevent the formation of fibrin clots.
  • ATIII antithrombin III
  • Heparin is one of the most potent anticoagulants used for treatment and prevention of deep vein thrombosis and pulmonary embolism. In vivo, however, applications of heparin are very limited. Because of its hydrophilicity and high negative charge, heparin is not absorbed efficiently from the GI tract, nasal or buccal mucosal layers, and the like. Therefore, the only routes of administration used clinically are intravenous and subcutaneous injections. Moreover, since heparin is soluble in relatively few solvents, it is hard to use for coating surfaces of medical devices or in delivery systems.
  • a bile acid such as deoxycholic acid or glycocholic acid
  • a hydrophobic agent such as cholesterol, or an alkanoic acid.
  • compositions comprising heparin covalently bonded to a hydrophobic agent selected from the group consisting of bile acids, sterols, and alkanoic acids, and mixtures thereof.
  • the composition can also include a pharmaceutically acceptable carrier.
  • the hydrophobic agent is a bile acid selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixtures thereof, and the like.
  • the hydrophobic agent is a sterol selected from the group consisting of cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol, and mixtures thereof, and the like.
  • the hydrophobic agent is an alkanoic acid comprising about 4 to 20 carbon atoms.
  • Preferred alkanoic acids include butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, and mixtures thereof, and the like.
  • the heparin comprises a molecular weight of at least about 3000, and more preferably at least about 6000. In certain preferred embodiments, the heparin comprises a molecular weight less than about 12,000.
  • the macromolecular agent is a member selected from the group consisting of heparin, heparan sulfate, sulfonyl polysaccharide, heparinoids, polysaccharide derivatives, and mixtures thereof, and the like.
  • the macromolecular agent is a peptide, such as insulin or calcitonin.
  • FIGS. 5A-P show micrographs of hematoxylin and eosin stained gastrointestinal tissues that were isolated from rats after oral administration of 100 mg/kg of heparin-DOCA conjugate:
  • FIGS. 5A-D show cross sections of the stomach after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 5E-H show cross sections of the duodenum after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 5I-L show cross sections of the jejunum after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 5M-P show cross sections of the ileum after 0, 1, 2, and 3 hours, respectively; the original magnification was 100 ⁇ in all FIGS. 5A-P.
  • FIGS. 6A-P show electron micrographs of membrane or microvilli in gastrointestinal tissues isolated from rats after oral administration of 100 mg/kg of heparin-DOCA conjugate:
  • FIGS. 6A-D show cross sections of the of the stomach after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 6E-H show cross sections of the duodenum after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 6I-L show cross sections of the jejunum after 0, 1, 2, and 3 hours, respectively;
  • FIGS. 6M-P show cross sections of the ileum after 0, 1, 2, and 3 hours, respectively; the original magnification was 25,000 ⁇ in all of FIGS. 6A-P.
  • FIGS. 7A and 7B show clotting time profiles (FIG. 7A) and concentration profiles (FIG. 7B) of heparin-DOCA conjugates after oral administration in rats: ⁇ —LMWH(3K)-DOCA; ⁇ —LMWH(6K)-DOCA; ⁇ —heparin-DOCA (also referred to herein as UFH-DOCA).
  • FIGS. 8A and 8B show clotting time profiles (FIG. 8A) and concentration profiles (FIG. 8B) of LMWH(6K)-DOCA after oral administration in rats: ⁇ —20 mg/kg of LMWH(6K) control; ⁇ —100 mg/kg of LMWH(6K) control; ⁇ —20 mg/kg LMWH(6K)-DOCA; ⁇ —50 mg/kg LMWH(6K)-DOCA; ⁇ —100 mg/kg LMWH(6K)-DOCA.
  • a bile acid includes a mixture of two or more of such bile acids
  • an alkanoic acid includes reference to one or more of such alkanoic acids
  • reference to “a sterol” includes reference to a mixture of two or more sterols.
  • Bile acids means natural and synthetic derivatives of the steroid, cholanic acid, including, without limitation, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixtures thereof, and the like.
  • sterols means alcohols structurally related to the steroids including, without limitation, cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol, and mixtures thereof, and the like.
  • alkanoic acids means saturated fatty acids of about 4 to 20 carbon atoms.
  • Illustrative alkanoic acids include, without limitation, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, and mixtures thereof, and the like.
  • hydrophobic heparin derivative and “amphiphilic heparin derivative” are used interchangeably.
  • Heparin is a very hydrophilic material. Increasing the hydrophobicity of heparin by bonding a hydrophobic agent thereto results in what is termed herein an amphiphilic heparin derivative or hydrophobic heparin derivative. Either term is proper because the heparin derivative has increased hydrophobicity as compared to native heparin and the heparin derivative has a hydrophilic portion and a hydrophobic portion and is, thus, amphiphilic.
  • aPTT activated partial thromboplastin time
  • FXa means factor Xa
  • DOCA deoxycholic acid
  • heparin-DOCA means a conjugate of heparin and deoxycholic acid
  • macromolecule means polypeptide, polysaccharide, and nucleic acid polymers with a molecular weight typically greater than 1000.
  • peptide means peptides of any length and includes proteins.
  • polypeptide and oligopeptide are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
  • Typical of peptides that can be utilized are those selected from group consisting of oxytocin, vasopressin, adrenocorticotrophic hormone, epidermal growth factor, prolactin, luliberin or luteinising hormone releasing hormone, growth hormone, growth hormone releasing factor, insulin, somatostatin, glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines, and synthetic analogues, modifications and pharmacological
  • an effective amount means an amount of a pharmacologically active agent that is nontoxic but sufficient to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio attending any medical treatment.
  • an effective amount of a heparin-DOCA conjugate is an amount sufficient to provide a selected level of anticoagulation activity.
  • Heparin is used as an antithrombogenic agent to prevent blood coagulation.
  • Heparin is highly hydrophilic because of a high density of negative charges such as are provided by sulfonic and carboxylic groups. Due to this hydrophilicity, heparin is usually administered by intravenous or subcutaneous injection.
  • Heparin derivatives with slightly hydrophobic properties or amphiphilic properties and with high bioactivity are described herein.
  • Hydrophobic agents such as bile acids, e.g. deoxycholic acid (DOCA); sterols, e.g. cholesterol; and alkanoic acids, e.g. lauric acid and palmitic acid, were coupled to heparin.
  • bile acids e.g. deoxycholic acid (DOCA)
  • sterols e.g. cholesterol
  • alkanoic acids e.g. lauric acid and palmitic acid
  • Both deoxycholic acid and cholesterol are non-toxic since they are naturally occurring compounds found in the body.
  • the amine groups of heparin were coupled with carboxyl groups of the hydrophobic agents.
  • the end carboxylic groups in DOCA, lauric acid, and palmitic acid were used directly for the coupling reaction, while the hydroxy group of cholesterol was activated by reaction with chloroacetic acid before coupling. It was determined that conjugating such hydrophobic moieties to the amine groups of heparin had little or no effect on heparin bioactivity.
  • the coupling between heparin and hydrophobic agents was confirmed by detecting the resulting amide bond by FT-IR and 13 C-NMR analysis.
  • the yield of the coupling reaction was about 70 to 80% and was not significantly changed by changing the hydrophobic agents or feed molar ratios.
  • the amount of DOCA in the conjugate was also increased.
  • the weight % of DOCA in heparin-DOCA was 24% when the feed molar ratio of heparin to DOCA was 1:200. This molar ratio was very high compared to the ratio of amine groups in heparin to DOCA. Therefore, this feed ratio is estimated as an excess amount of DOCA.
  • the hydrophobic heparin derivatives according to the present invention would have many medical applications.
  • the hydrophobic heparin can be administered orally.
  • the oral administration of heparin can greatly extend the usage of heparin as an oral anti-coagulant drug.
  • the heparin derivative is formulated with a pharmaceutically acceptable carrier such as is well known in the art.
  • hydrophobic heparin derivatives can be used as a coating material for medical devices such as catheters, cardiopulmonary bypass circuits, heart lung oxygenators, kidney dialyzers, stent or balloon coating for preventing restenosis, and the like.
  • the hydrophobic heparin derivative is typically mixed with a carrier, and then coated on the surface of the medical device by a film casting technique such as is well known in the art.
  • heparin-hydrophobic agents were also found to have a tendency in fast protein liquid chromatography (FPLC®) to exhibit hydrophobic interactions with hydrophobic media, as shown by chromatography on Phenyl Sepharose® (eluting in ammonium sulfate buffer rather than phosphate buffer). These heparin derivatives showed enhanced binding affinity when compared to unmodified heparin. The increased interaction of modified heparin derivatives with Phenyl Sepharose® is attributable to its enhanced hydrophobicity, the result of the hydrophobic functional groups present. These results suggest hydrophobic heparin can be obtained by conjugating a bile acid, sterol, or alkanoic acid to heparin. In solubility tests, polar solvents or organic solvents were suitable to dissolve the heparin-hydrophobic agent conjugates.
  • FPLC® fast protein liquid chromatography
  • the heparin-deoxycholic acid conjugate showed good solubility in 65% acetone solution (35% water).
  • bioactivity of modified heparin derivatives was not appreciably influenced by conjugation with hydrophobic agents.
  • the role of a hydrophobic agent conjugated to heparin was studied with respect to two biological activities of heparin as determined by anticoagulation and factor Xa assays. Although hydrophobicity is associated with a somewhat reduced anticoagulant activity and antifactor Xa activity, the decrease of bioactivity was not considered serious.
  • bioactivity of heparin in heparin-hydrophobic agent conjugates exhibited a progressive reduction, however, when the amount of hydrophobic agent in the conjugate exceeded 20 wt. %. At less than 20 wt. % of hydrophobic agent in the conjugates, the bioactivity of the conjugates was greater than 80% of the bioactivity of unmodified heparin. It is suggested that 80% of bioactivity in hydrophobic heparin is enough to support bioactivity in medical applications.
  • the resulting solution was reacted for 5 hours at room temperature under vacuum, and then the byproduct dicyclohexylurea (DCU), which precipitated during the reaction, was removed.
  • the unreacted DCC was removed by adding a drop of distilled water and filtering.
  • the remaining HOSu was also removed by adding 15 ml of distilled water.
  • the activated DOCA was precipitated and then lyophilized.
  • the activated DOCA was then dissolved in DMF and reacted with heparin for 4 hours at room temperature.
  • the amounts of heparin used in such reactions ranged from 40 to 400 mg. After reaction, there were two types of products: a water soluble product and a water-insoluble product.
  • the synthesized heparin-DOCA was further purified by reverse phase chromatography.
  • a phenyl-Sepharose CL-4B column (HR 16/30 I.D.) was washed with 100 ml of distilled water, 40 ml of 50 mM phosphate buffer (pH 7.0), 40 ml of 50 mM phosphate buffer (pH 7.0) containing 1.7 M ammonium sulfate, and 40 ml of 50 mM phosphate buffer, respectively.
  • Five milliliters of the heparin-DOCA solution (1 mg/ml) was loaded in the column and the heparin-DOCA was fractionated by step elution with an ammonium sulfate solution.
  • heparin derivatives prepared according to this procedure were characterized by FT-IR and NMR according to methods well known in the art to prove the successful coupling between heparin and the hydrophobic agent.
  • heparin-DOCA heparin-cholesterol and heparin-alkanoic acid prepared according to the procedures of Examples 1-3
  • the production yield, molecular weight, and binding mole ratios between heparin and hydrophobic agents varied according to the mole ratio of reactants.
  • the yield of heparin-DOCA conjugates was in the range of 71 to 77%.
  • the amount of hydrophobic agent in modified heparin derivatives was calculated by subtracting the molecular weight of heparin (i.e., 12,386 daltons as determined by light scattering) from the measured molecular weight of each heparin derivative.
  • the amount of DOCA in heparin-DOCA conjugates was increased from 7 to 24%.
  • the yield also was in the range from 73 to 78%.
  • the amount of cholesterol in such hydrophobic heparin conjugates was slightly lower than the amount of DOCA in heparin-DOCA conjugates.
  • heparin-lauric acid and heparin-palmitic acid conjugates similar amounts of alkanoic acid were coupled to heparin.
  • Anticoagulant activities of heparin derivatives were determined by aPTT assay and FXa chromogenic assay. The activities of heparin derivatives in the prevention of fibrin clot formation were measured by aPTT assay.
  • Each of the platelet-poor-plasma containing heparin standards (0.1 to 0.7 U/ml, 0.1 ml) and plasma samples containing heparin derivatives (0.1 ml) was incubated with 0.1 ml of aPTT reagent for 2 min at 37° C. After the incubation, 0.1 ml of 0.02 M calcium chloride was added, and the time was recorded from this point until the fibrin clot was formed.
  • the bioactivity of the heparin derivative was calculated by comparing the clotting time with the heparin standard curve. The clotting time was linearly proportional to the activity of heparin in the plasma.
  • the activity and the concentration of heparin derivatives were also determined by FXa chromogenic assay.
  • Each of the heparin standards and plasma samples containing heparin derivatives 25 ⁇ l was mixed with 200 ⁇ l of AT III solution (0.1 IU/ml), where the ATIII concentration was in excess of the heparin concentration.
  • This solution was incubated at 37 for 2 min, and 200 ⁇ l of FXa (4 nkcat/ml) was added. The resulting solution was then incubated for an additional 1 min.
  • the concentration of FXa was also in excess of the heparin concentration.
  • FXa substrate 200 ⁇ l, 0.8 ⁇ mol/ml was then added and incubated at 37 for 5 min. The reaction was terminated by adding 200 ⁇ l of acetic acid (50% solution).
  • the bioactivity and the concentration of heparin in the plasma sample were calculated from the absorbance at 405 nm.
  • Heparin-DOCA Oral Administration of Heparin-DOCA.
  • Sprague-Dawley rats male, 250-260 g
  • the rats were anesthetized with diethyl ether and then were administered a single dose of heparin derivative through an oral gavage that was carefully passed down the esophagus into the stomach.
  • the gavage was made of stainless steel with a blunt end to avoid causing lesions on the tissue surface.
  • the solution containing the heparin derivative was prepared in a sodium bicarbonate buffer (pH 7.4).
  • the total administered volume of heparin-derivative-containing solution was 0.3 ml.
  • the dose amount was varied at 50, 80, 100, and 200 mg/kg, respectively.
  • the absorption of heparin-DOCA in the GI tract was determined according to the dose amount in the range of 50 to 200 mg/kg.
  • the mole ratio of coupled DOCA to heparin in the heparin-DOCA conjugate was 10.
  • the clotting time measured by aPTT assay, was about 18 seconds and this value did not change over time.
  • the average value of the baseline was 18 seconds, indicating that the raw heparin was not absorbed in the GI tract.
  • the aPTT value was about 20 seconds, and this value did not change over time.
  • the clotting time at one hour was linearly increased with the increase of dosage.
  • the clotting times at one hour were 25.8 ⁇ 2.6, 43.1 ⁇ 4.0, 51.2 ⁇ 9.3, and 136 ⁇ 33 seconds, respectively.
  • the clotting time at one hour increased greatly, above 7-times the baseline.
  • the heparin-DOCA conjugate greatly enhanced the absorption of heparin in the GI tract, in contrast to DOCA mixed with heparin in a physical mixture, which did not enhance heparin absorption.
  • the concentration of heparin-DOCA conjugate in the plasma was determined by FXa assay, as shown in FIG. 2 .
  • the concentration profiles of heparin-DOCA conjugate over time were similar to the results of the aPTT assay shown in FIG. 1 .
  • the concentration of absorbed heparin-DOCA increased with the increase of the dosage.
  • the therapeutic target range was 0.1 to 0.2 IU/ml.
  • the mean concentration peak at one hour was about 9-10 times the baseline and the concentration at that time was about 1.0 IU/ml.
  • the plasma concentration of heparin-DOCA conjugate returned to the baseline after 3 hours. Therefore, the absorption of heparin-DOCA in the GI tract was confirmed.
  • Heparin-DOCA Conjugate Absorption in the GI Tract of Rats To determine the absorption of heparin-DOCA conjugate in the GI tract as a function of the ratio of DOCA to heparin, heparin-DOCA conjugates were synthesized with DOCA:heparin mole ratios of 2.5, 5.0, and 10.0, as described in Example 1. As shown in Table 1, the bioactivity of heparin-DOCA conjugates decreased slightly as the mole ratio of DOCA to heparin increased. However, since the molecular weight of heparin-DOCA increased as the mole ratio of DOCA to heparin increased, the bioactivity of heparin-DOCA conjugates as a function of mole ratio decreased only about 5%. That is, the bioactivities of heparin and heparin-DOCA conjugate (10:1 mole ratio) were 1,734 and 1,632 ⁇ 7 IU/mol, respectively.
  • FIG. 3 shows the change in the clotting time according to the coupled mole ratio of DOCA to heparin.
  • the dosage of heparin-DOCA conjugate was 100 mg/kg.
  • the bioactivity of heparin-DOCA conjugate slightly decreased, as shown in Table 1, whereas the maximum clotting time increased. This result indicates that the heparin-DOCA conjugate facilitated absorption of heparin in the GI tract of rats.
  • heparin-DOCA conjugate Even though cholesterol is more hydrophobic than DOCA, however, heparin-DOCA conjugate exhibited a higher clotting time than heparin-cholesterol conjugate. Possible explanations for this observation include (1) the amphiphilic properties of heparin-DOCA conjugate, which may improve the permeability of the heparin derivative in the GI wall, and (2) the interaction between the DOCA moiety of the heparin-DOCA conjugate and the DOCA receptors in the GI wall, especially in the ileum, which might increase the adhesion of heparin-DOCA conjugate to the GI wall, thereby increasing the probability of absorption.
  • heparin-DOCA conjugate was administered to rats by oral gavage according to the procedure of Example 10.
  • the mole ratio of coupled DOCA to heparin in the heparin-DOCA conjugate was 10. That is, ten molecules of DOCA were coupled to one molecule of heparin. The dose amount was 200 mg/kg.
  • rats were anesthetized with diethyl ether and were sacrificed by cutting the diaphragm. Gastric, duodenal, jejunal, and ileal tissues were removed from the rats and fixed in neutral buffered formalin for processing. GI tissues sampled before administration of heparin-DOCA conjugate were prepared as control samples.
  • the tissue specimens were washed with alcohol to remove any water. Specimens were perfused with colored silicone and embedded in paraffin. The embedded specimens were cut into 5 ⁇ m sections using a microtome at ⁇ 20° C., and picked up on a glass slide. The tissue sections were then washed with xylene and absolute alcohol, respectively, to remove the paraffin. The prepared 5 ⁇ m sections were then stained with hematoxylin and eosin (H&E) according to procedures well known in the art. At least 4 rats were used for each treatment.
  • H&E hematoxylin and eosin
  • TEM transmission electron microscopy
  • the gastric, duodenal, jejunal, and ileal tissues were fixed with 1% osmium tetroxide in PBS (0.1 M, pH 7.4), and then hydrated by changing the alcohol concentration gradually from 50 to 100%.
  • the hydrated tissues were infiltrated with propylene oxide and embedded with an epon mixture.
  • the embedded tissues were sectioned as about 50-60 nm thickness slides. These slides were stained very lightly with uranyl acetate and lead citrate for 1 minute, and were observed by TEM (Hitachi 7100, Tokyo, Japan).
  • FIGS. 5A-P show that there was no evidence of damage to the GI wall, such as occasional epithelial cell shedding, villi fusion, congestion of mucosal capillary with blood, or focal trauma, in any parts of the stomach, duodenum, jejunum, or ileum. These results confirm that increased absorption of heparin derivatives was not caused by the disruption of the gastrointestinal epithelium.
  • FIGS. 6A-P show the electron microscopic morphology of microvilli after exposure to heparin derivatives.
  • the control samples showed healthy tight junctions, microvilli, and mitochondria. After 1, 2, and 3 hours, the cell appearance in all sections showed no signs of damage, such as microvilli fusion, dissolution, disoriented cell layer with porosity, or cytotoxic effect.
  • Microvilli exposed to heparin derivatives were also found to be as healthy as the control. The absence of tissue damage indicates that the enhancing effect of the coupled DOCA on heparin absorption in the GI tract was not caused by changing the tissue structure.
  • Conjugation of Lower Molecular Weight Heparin to DOCA Conjugates of heparin to DOCA were synthesized according to the procedure of Example 1 except that unfractionated heparin (“UFH”), i.e., the compound referred to simply as “heparin” in previous examples, 6000 molecular weight heparin (“LMWH(6K)”), and 3000 molecular weight heparin (“LMWH(3K)”) were used.
  • UHF unfractionated heparin
  • LMWH(6K) 6000 molecular weight heparin
  • LMWH(3K) 3000 molecular weight heparin
  • the maximum ratio of DOCA to heparin obtained in UFH-DOCA was 10 when the feed ratio of UFH to DOCA was 1:200. Under these conditions, the ratios obtained with lower molecular weight heparins were 1.3 for LMWH(3K)-DOCA and 3.6 for LMWH(6K)-DOCA.
  • the mole ratio of DOCA to heparin decreased with the decrease in molecular weight of heparin because of the fewer number of amine groups available for bonding to DOCA.
  • Bioactivities of the heparin-DOCA conjugates also decreased with the decrease of molecular weight of heparin, although all heparin-DOCA conjugates demonstrated similar bioactivities in the range of 116.9 ⁇ 1.6 to 134.3 ⁇ 0.8 by FXa assay. After conjugation with DOCA, all of the heparin-DOCA conjugates showed above 70% relative bioactivity compared to the unmodified heparin.
  • FIG. 7 shows the effect of molecular weight of heparin on the absorption of heparin-DOCA conjugates in the GI tract.
  • LMWH(3K)-DOCA, LMWH(6K)-DOCA, and UFH-DOCA were each administered by oral gavage at 100 mg/kg dosage.
  • the clotting times of LMWH(3K)-DOCA and UFH-DOCA were lower than that of LMWH(6K)-DOCA; the mean aPTT times at 1 hour were 31.0 ⁇ 6.01 and 51.0 ⁇ 8.7, respectively (p ⁇ 0.005).
  • the concentration profiles of heparin-DOCA conjugates with time were similar to the results of the aPTT assay. When UFH-DOCA was administered at 100 mg/kg dosage, the peak concentrations of plasma was 4.10 ⁇ 1.3 ⁇ g/ml, which was very low compared to the concentration of LMWH(6K)-DOCA at the same dosage level.
  • the absorption of LMWH(6K)-DOCA in the GI tract was determined according to the dose amount in the range of 20 to 100 mg/kg, as shown in FIG. 8 .
  • the clotting time as measured by aPTT assay was about 30 seconds at 1 hour after dosing. This curve fell to baseline at 2 hours after dosing.
  • oral delivery of LMWH(6K)-DOCA resulted in the increased heparin absorption in rats as shown by the highly elevated aPTT values.
  • LMWH(6K)-DOCA When LMWH(6K)-DOCA was dosed at 100 mg/kg, the peak plasma aPTT value was about 87.8 ⁇ 11.1 seconds (the baseline aPTT values averaged 20 seconds). Heparin derivatives dosed at 20 mg/kg and 50 mg/kg gave mean peak aPTT responses of 52.5 ⁇ 4.7 and 68.4 ⁇ 7.2 seconds, respectively (p ⁇ 0.005).
  • the therapeutic range of heparin which is about 1.5-2.5 times baseline in aPTT, is matched with a dose of 20 mg/kg, as shown in FIG. 8 A.
  • Concentrations of heparin derivatives in the plasma could be determined using the anti-FXa assay.
  • concentration of LMWH(6K)-DOCA was 1.34 ⁇ 0.28 ⁇ g/ml.
  • the low concentration of LMWH(6K) in the plasma could not facilitate anticoagulation activity.
  • the maximum peak of LMWH(6K)-DOCA was 8.21 ⁇ 1.6 ⁇ g/ml at a dose of 100 mg/kg, as shown in FIG. 8 B.
  • the therapeutic target range was 0.1 to 0.2 IU/ml.
  • the mean concentration peaks were about 9-10 times the baseline.
  • GI tract tissues from rats given a single dose of 100 mg/kg of lower molecular weight heparin-DOCA conjugates prepared according to the procedure of Example 14 were examined histologically according to the procedures of Example 13. The results were substantially similar to those of Example 13. That is, no evidence of damage to any of the tissues of the GI wall were detected.

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